CURRENT OBJECTIVES:[unreadable] [unreadable] 1) To identify endogenous factors that modulate mast cell reactivity to antigen: this year we have completed studies on PGE2. Like adenosine, receptors for PGE2 are expressed on mast cells, PGE2 accumulates in inflamed tissues, and by itself minimally stimulates mast cells. We have also examined another potential co-stimulant, IL-33. IL-33 is a recently discovered member of the IL-1 super family that acts through an orphan receptor, ST2, to activate TLR-mediated signaling pathways. Of particular interest, IL-33 accumulates in inflamed tissues associated with allergic and autoimmune diseases. The question addressed is whether IL-33, like TLR ligands, acts in synergy with antigen and if so by what mechanism.[unreadable] [unreadable] 2) To continue studies of the mechanisms by which glucocorticoids suppress activation of mast cells. In the current period we have examined the mechanism of upregulation of glucocorticoid-inducible inhibitory regulators at a transcriptional level.[unreadable] [unreadable] 3) To continue studies of the inhibitory actions of glucocorticoids on the activation of mast cells by TLR ligands. Of specific interest is the upregulation of suppressor of cytokine signaling1 (Socs1) and IKKalpha by dexamethasone. [unreadable] [unreadable] RESULTS:[unreadable] [unreadable] 1) IDENTIFICATION OF FACTORS THAT MODULATE RESPONSES TO ANTIGEN: In collaboration with Gilfillan and Metcalfe (LAD, NIAID) we found that PGE2, acting through its G protein-coupled EP3 receptor, markedly potentiated FceRI-mediated activation of phospholipase (PL) C, calcium signal, calcium-dependent PKC isoforms, and degranulation. Of note, activation of both EP3-linked PLCbeta and FceRI-linked PLCgamma were amplified by co-stimulation to reveal unexpected cross talk between these two PLCs (ref. 1). These and our previous studies with adenosine, Kit ligand (SCF) and TLR ligands, illustrate the remarkable ability of co-stimulants to augment mast cells to antigen. In fact, simultaneous inhibition of SCF (an essential growth factor for mast cells) and antigen mediated signaling events with hypothemycin is a particularly effective means of suppressing mast cell activation (ref. 2). An incidental finding some years ago was that the plant flavanoid, curcumin, was a potent inhibitor of mast cell cytokine production in vitro. A former post doctoral fellow of the PI has since continued studies with this compound and found that it is equally potent in suppressing passive cutaneous anaphylaxis (PCA) in vivo and acts at the level of Syk, a key kinase in the FceRI signaling pathways (ref. 3). [unreadable] [unreadable] IL-33 was found to be the most potent agent examined to date with respect to its ability to augment responses to antigen. At almost homeopathic concentrations (10 pg/ml), IL-33 markedly potentiated antigen-induced cytokine production in primary and tumor mast cell lines. This phenomenon was observed with concentrations of antigen that minimally stimulated cytokine production and degranulation. In effect, IL-33 finely tuned the mast cell response towards cytokine production. IL-33 was ineffective in MyD88-deficient mast cells indicating that IL-33 acted through TLR signaling pathways for which MyD88 is a critical adaptor protein. Preliminary evaluation of signaling mechanisms indicated that IL-33 robustly stimulated the NFkappB pathway and antigen the Ca2+/calcineurin/NFAT pathway which together produced a duality in signaling that was not evident with the individual stimulants. Therefore, production of IL-33 in inflamed tissue may profoundly affect mast cell cytokine production in response to antigen and perhaps other stimulants.[unreadable] [unreadable] 2) UPREGULATION OF INHIBITORY REGULATORS BY GLUCOCORTOIDS, A NOVEL MECHANISM OF ACTION OF GLUCORTICOIDS: The participation of GR and GRE in some inhibitory actions of dexamethasone in mast cells was verified by overexpression of a mutated GR that cannot interact with GREs. In such cells the ability of dexamethasone to inhibit degranulation and release of arachidonic acid was reduced without affecting its ability to suppress of cytokine production. This finding is consistent with the notion that suppression of cytokine production is due to "transrepression" of cytokine gene transcription which would not involve GRE whereas the upregulation of inhibitory regulators might involve "transactivation" via GRE (see Goals and Objectives section). The upregulation of the inhibitory regulators Dok1, SLAP1, and DUSP1 in the RBL-2H3 mast cell line was inhibited by actinomycin D and was thus dependent on gene transcription. Examination of the gene sequences by the MULAN program revealed a half GRE and a full GRE as potential regulators of the Dok1 and SLAP1 genes, respectively. As indicated by luciferase reporter assays, the SLAP1 GRE regulated gene transcription in dexamethasone treated cells whereas the half GRE of Dok1 did not do so. Binding of GR to the SLAP1 GRE was confirmed by chromatin immunoprecipitation assay. Similar studies with various steroids and a GR antagonist verified the GR/SLAP1 GRE connection. Therefore, upregulation of SLAP1 by dexamethasone is regulated primarily through interaction of GR with GRE while other transcription mechanisms come into play for the upregulation of Dok1 and DUSP1. Our findings add a new dimension to our understanding of the inhibitory actions of glucocorticoids. These findings are relevant to the current development of "dissociated" steroids that lack the "transactivation" potential of clinically approved glucococorticoids, a potential that was previously thought to account for the undesirable side-effects of glucocortoids. However, glucocorticoids lacking this "transactivation" potential may also fail to induce inhibitory regulators such as those discovered in our studies (manuscript submitted). [unreadable] [unreadable] 3) SYNERGY BETWEEN ANTIGEN AND TLR LIGANDS IS SUPPRESSED BY GLUCOCORTICOIDS: The enhanced production of cytokines on co-stimulation of mast cells with antigen and TLR ligands was attributed to the engagement of a more effective repertoire of transcription factors for cytokine gene transcription (Blood 107:610, 2006 and see previous reports). Glucocorticoids potently inhibit production of cytokines in response TLR ligands alone or in combination with antigen. As with antigen stimulation, the inhibitory effects were apparent after a delay of several hours on exposure to low concentrations of dexamethasone and were mediated via GR. Again in common with antigen stimulation, activation of MAP kinases by TLR ligands is inhibited by dexamethasone possibly through the induction of DUSP1 and Dok1. In addition, the activation of upstream TAK1 by TRAF6 and downstream NFkappB are key targets for dexamethasone in TLR activated mast cells. Use of chip arrays and RT-PCR revealed several additional dexamethasone-inducible inhibitory regulators, SOCS1, its cognate CIS, and IKKalpha. SOCS1 but not CIS was found to associate with TRAF6 and enhance its degradation. Knockdown of SOCS1, partially reverses the inhibitory effects of dexamethasone on TRAF6 degradation and phosphorylation of TAK1 to suggest that dexamethasone may act at this level through upregulation of SOCS1. Levels of IKKalpha, a negative regulator of NFkappaB, are increased more than 10 fold in dexamethasone treated cells. For this reason, IKKalpha is being studied as a fifth candidate for mediating the inhibitory actions of glucocorticoids. The ability of glucocorticoids to suppress both antigen and TLR-mediated signaling events suggests that they may be especially effective when allergic disease is exacerbated by viral or bacterial infection (ref. 4)